Single-cell analysis reveals the loss of FABP4-positive proliferating valvular endothelial cells relates to functional mitral regurgitation

Abstract

Background: Functional mitral regurgitation (MR) is a common form of mitral valve dysfunction that often persists even after surgical intervention, requiring reoperation in some cases. To advance our understanding of the pathogenesis of functional MR, it is crucial to characterize the cellular composition of the mitral valve leaflet and identify molecular changes in each cell subtype within the mitral valves of MR patients. Therefore, we aimed to comprehensively examine the cellular and molecular components of mitral valves in patients with MR.

Methods: We conducted a single-cell RNA sequencing (scRNA-seq) analysis of mitral valve leaflets extracted from six patients who underwent heart transplantation. The cohort comprised three individuals with moderate-to-severe functional MR (MR group) and three non-diseased controls (NC group). Bioinformatics was applied to identify cell types, delineate cell functions, and explore cellular developmental trajectories and interactions. Key findings from the scRNA-seq analysis were validated using pathological staining to visualize key markers in the mitral valve leaflets. Additionally, in vitro experiments with human primary valvular endothelial cells were conducted to further support our results.

Results: Our study revealed that valve interstitial cells are critical for adaptive valve remodelling, as they secrete extracellular matrix proteins and promote fibrosis. We discovered an abnormal decrease in a subpopulation of FABP4 (fatty acid binding protein 4)-positive proliferating valvular endothelial cells. The trajectory analysis identifies this subcluster as the origin of VECs. Immunohistochemistry on the expanded cohort showed a reduction of FABP4-positive VECs in patients with functional MR. Intervention experiments with primary cells indicated that FABP4 promotes proliferation and migration in mitral valve VECs and enhances TGFβ-induced differentiation.

Conclusions: Our study presented a comprehensive assessment of the mitral valve cellular landscape of patients with MR and sheds light on the molecular changes occurring in human mitral valves during functional MR. We found a notable reduction in the proliferating endothelial cell subpopulation of valve leaflets, and FABP4 was identified as one of their markers. Therefore, FABP4 positive VECs served as proliferating endothelial cells relates to functional mitral regurgitation. These VECs exhibited high proliferative and differentiative properties. Their reduction was associated with the occurrence of functional MR.

Keywords: FABP4; Functional mitral regurgitation; ScRNA-seq; Valvular endothelial cell.

Fig. 1

Clustering and identification of cell types in nondiseased and functional regurgitated human mitral valves with scRNA-seq data. A Six mitral valves from three normal controls and three patients with MR were harvested, and scRNA-seq was separately performed. B The UMAP plot of the combined six samples shows clusters, sample types, individual patients, and cell types. C The gene markers and each cell type identified are shown in the violin plot. D Dot plot of the top 5 marker genes in VIC clusters. The dot size corresponds to the proportion of cells within the group expressing each gene, and the dot color corresponds to the expression level. E The number of cells measured in six specimens. F The composition of each cell type in the NC and MR groups. G Box plots comparing the proportions of each cell type between the two groups. MR, mitral regurgitation; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection; VIC, valvular interstitial cell; VEC, valvular endothelial cell

Fig. 2

Dynamic VIC cluster in human mitral valve leaflet. A A UMAP plot of all VIC cells coloured according to cluster. B Dot plot of the top 5 marker genes in VIC clusters. The dot size corresponds to the proportion of cells within the group expressing each gene, and the dot color corresponds to the expression level. C The enriched GO terms for each VIC cluster. D Heatmap of TF regulons in each VIC cluster. E The proportion of each VIC cluster in six samples. F Volcano plot showing the DEGs between the NC group and MR group in VIC. G The expression level and regulon activity of the TFs STAT1, EGR2, and SREBF1 in each VIC cluster. H Enrichment plots for representative pathways dysregulated in VIC in the MR. The vertical lines in the enrichment plot indicate the members of the gene set appear in the ranked list of genes. VIC, valvular interstitial cell; UMAP, uniform manifold approximation and projection; GO, Gene Ontology; MR, mitral regurgitation; TF, transcription factor; DEGs, differentially expressed genes

Fig. 3

Dynamic VECclusters in human mitral valve leaflet. A A UMAP plot of all VECs coloured according to cluster. B A UMAP plot of all VECs according to phase. C Dot plot of the top 5 marker genes in VEC clusters. The dot size corresponds to the proportion of cells within the group expressing each gene, and the dot colour corresponds to the expression level. D The enriched GO terms of each VEC cluster. E SCENIC analysis of the expression of different TF regulons between each cluster. The data are coloured according to their expression levels. F The expression level and regulon activity of the TFs GATA6 and FOXF in each VEC cluster. G Differential gene expression of VECs in the NC group and MR group is shown in a volcano plot. H Comparison of the percentage of each cell type among the three clusters of VECs. I The enrichment scores of four different biological processes in each VEC subcluster. J Pseudotime single-cell trajectory reconstructed by slingshot for VEC. K Plot of marker and functional genes along the VEC2 trajectories. VEC, valvular endothelial cell; UMAP, uniform manifold approximation and projection; GO, Gene Ontology; MR, mitral regurgitation; TF, transcription factor; DEGs, differentially expressed genes

Fig. 4

Dynamicmyeloid cluster in the human mitral valve leaflet. A A UMAP plot of all myeloid cells coloured according to cluster. B Dot plot of the top 5 marker genes in myeloid clusters. The dot size corresponds to the proportion of cells within the group expressing each gene, and the dot colour corresponds to the expression level. C Heatmap of inflammatory cytokines, chemokines, and HLA in each cluster. D UMAP of myeloid canonical markers. E The enriched GO terms of each myeloid cluster. F SCENIC analysis of the expression of different TF regulons between each cluster. The data are coloured according to their expression levels. G The expression level and regulon activity of the TF BRCA1 in each myeloid cluster. H Comparison of the percentage of each cell type among the five clusters of myeloid cells. I Differential gene expression of myeloid cells in the NC group and MR group is shown in a volcano plot

Fig. 5

Dynamiclymphocyte clusters in the human mitral valve leaflet. A A UMAP plot of all lymphocyte cells coloured according to cluster. B UMAP of canonical lymphocyte cell markers. C Dot plot of the top 5 marker genes in the lymphocyte clusters. The dot size corresponds to the proportion of cells within the group expressing each gene, and the dot colour corresponds to the expression level. D The enriched GO terms of each lymphocyte cluster. E Comparison of the percentage of each cell type among the five clusters of lymphocytes. F SCENIC analysis of the expression of different TF regulons between each cluster. The data are coloured according to their expression levels. G Differential gene expression of myeloid cells in the NC group and MR group is shown in a volcano plot

Fig. 6

Loss of proliferating endothelial cells in the endothelium associated with inadequate adaptation to regurgitation. A Immunohistochemical staining of FABP4 in NC and MR specimens. B Quantification of the FABP4^＋ cell ratio in the endothelium per image (n = 12 in Group A, 11 in Group B, and 10 in Group C). C ROC curve for the predictive ability of FABP4⁺ VECs on the severity of mitral valve regurgitation. D Immunohistochemistry results for CD34 in the NC and MR groups. E Differential expression of CD34 between the NC and MR groups in IHC. F Immunostaining of FABP4, CD34 and CD31. G Immunostaining of ACTA2 (αSMA) and vWF in the NC and MR groups. H Quantification of the percentage of positive cells per image (n = 3 per condition). Mean ± SEM, two-tailed t test. Group A indicates patients with no or mild MR; Group B indicates patients with moderate MR; Group C indicates patients with severe MR. ROC, receiver operating characteristic; NC, non-diseased control; MR, mitral regurgitation

Fig. 7

Extensive cell–cell interactions in the mitral valve. A Overall intercellular communication between each pair of cell populations in the comparison of NC and MR. The width of bands corresponding to the number of ligand‒receptor pairs. B Heatmaps of the number of interactions between NC and MR, showing the outgoing and incoming signalling in each cell group in greater detail (the top coloured bar plot represents the sum of each column of values displayed in the heatmap (incoming signalling). The right coloured bar plot represents the sum of each row of values (outgoing signalling). C Significant signalling pathways were ranked based on differences in the overall information flow within the inferred networks between NC and MR. The overall information flow of a signalling network was calculated by summarizing all communication probabilities in that network. D Circos plots showing the inferred intercellular communication network among VEC2 and other cell subtypes in the NC and MR groups. E Comparison of the specific significant ligand‒receptor pairs between the NC and MR groups, which contribute to signalling among VEC2 and the subclusters of VEC and VIC types. The dot colour reflects the communication probability, and the dot size represents the computed p value. An empty space means that the communication probability is zero. p values were computed from a one-sided permutation test

Fig. 8

The regulatory role of FABP4⁺ VECs in VEC proliferation, migration, and EndMT. A Representative images of human VECs from the cell scratch assay. B Quantitative analysis of the results of the cell scratch assay. C Representative images of human VECs from Ki-67 proliferation assay. D Quantitative analysis of the results of Ki-67 proliferation assay. E Representative images of EdU staining. F Quantitative analysis of the results of EdU staining. G Representative images of Western blotting. H Quantitative analysis of the results of Western blotting. A Scale bar, 500 μm. C Scale bar, 50 μm. E Scale bar, 100 μm. Points in each group in B, D, F, H represent 7, 30, 6, and 10 biological replicates, respectively. The data were normally distributed and had equal variance. One-way ANOVA followed by Tukey’s multiple comparison test was used for multiple groups. A value of P<0.05 was considered statistically significant